Nonacetylated α-tubulin was obtained from HeLa S3 cells (American Type Culture Collection, CCL-2.2TM) for in vitro acetylation assays or from WT and Atat1 KO mouse brains for in vitro transport assay according to the protocol adapted from (45). Cells were lysed in BRB80 [80 mM K Pipes (pH 6.8), 1 mM MgCl2, and 1 mM EGTA] supplemented with 1 mM β-mercaptoethanol, 1 mM PMSF, and protease inhibitors at 4°C. The soluble fraction was obtained by ultracentrifugation, followed by tubulin polymerization at 30°C min by adding 1 mM guanosine triphosphate (GTP) and 30% glycerol. MTs were pelleted by ultracentrifugation at 30°C for 30 min and depolymerized in BRB80 at 4°C; soluble tubulin was clarified by ultracentrifugation at 4°C. The second polymerization round was performed for 30 min at 30°C in the presence of high-molarity Pipes to remove mitogen-activated proteins; the MT pellet was sedimented by ultracentrifugation. MTs were then depolymerized in BRB80 at 4°C, and soluble tubulin was clarified and snap-frozen.

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